PEITC induces apoptosis of Human Brain Glioblastoma
GBM8401 Cells through the extrinsic- and intrinsic
-signaling pathways
Yu-Cheng Choua,b,c,d, Meng-Ya Changb, Mei-Jen Wangb, Tomor Harnode, Chih-Huang
Hungb, Hsu-Tung Leea,f, Chiung-Chyi Sheng, Jing-Gung Chung,h,i,*
aDivision of Neurosurgical Oncology, Neurological Institute, Taichung Veterans
General Hospital, Taichung 407, Taiwan
bInstitute of Medical Sciences, Tzu Chi University, Hualien 970, Taiwan cSchool of Medicine, National Defense Medical Center, Taipei 114, Taiwan
dRong Hsing Research Center for Translational Medicine, National Chung Hsing
University
eDepartment of Neurosurgery, Buddhist Tzu Chi General Hospital, College of
Medicine, Tzu Chi University, Hualien 970, Taiwan
fGraduate Institute of Medical Sciences, National Defense Medical Center, Taipei
114, Taiwan
gDivision of Minimally Invasive Skull Base Neurosurgery, Neurological Institute,
Taichung Veterans General Hospital, Taichung 970, Taiwan
hDepartments of Biological Science and Technology, China Medical University,
Taichung 404, Taiwan, R.O.C.
iDepartment of Biotechnology, Asia University, Taichung 413, Taiwan, R.O.C.
Running title: PEITC induces apoptosis in GBM 8401
*Correspondence to: Jing-Gung Chung, Department of Biological Science and Technology, China Medical University, No 91, Hsueh-Shih Road, Taichung 404, Taiwan. Tel: +886 4 2205 3366 ext 2161, Fax: +886 4 2205 3764, e-mail: [email protected]
ABSTRACT
Glioblastoma is the most common and most aggressive primary brain malignancy. The multimodality treatments for this tumor including surgery, radiotherapy, and chemotherapy, are still not completely satisfied. Phenethyl isothiocyanate (PEITC), one member of the isothiocyanate family, has been shown to induce apoptosis in many human cancer cells. In this study, we investigate the pro-apoptotic effects caused by PETIC in human brain glioblastoma multiforme GBM 8401 cells. In our data, PEITC induced the cell morphological changes and decreased the cell viability of GBM8401 cells in a dose- and time-dependent manner. Moreover, the analysis of cell cycle distribution detected by flow cytometry showed that PEITC induced significantly sub-G1 phase (apoptotic population) in GBM 8401 cells. In addition, PEITC promoted the production of reactive oxygen species (ROS) and Ca2+ release,
but decreased the mitochondrial membrane potential (ΔΨm) in treated cells. PEITC also induced caspases activities in GBM 8401 cells. Results from Western blot analysis indicated that PEITC promoted Fas, FasL, FADD, TRAIL, caspase-8, -9, -3, increased the pro-apoptotic protein (Bax, Bid and Bak), and inhibited the anti-apoptotic proteins (Bcl-2 and Bcl-xl) in GBM 8401 cells. Furthermore, PEITC promoted the release of cytochrome c, AIF and Endo G. GADD153, GRP 78, XBP-1 and IRE-1α, Calpain I and II in GBM 8401 cells. PEITC also promoted the expression of associated protein with endoplasmic reticulum (ER) stress. PEITC induces apoptosis through the extrinsic (death receptor) pathway, dysfunction of mitochondria, ROS induced ER stress, intrinsic (mitochondrial) pathway in GBM 8401 cells. The possible molecular mechanisms and signaling pathways of the anti-cancer properties of PEITC for human brain glioblastoma cells were postulated. Keyword: PEITC, apoptosis, caspases, mitochondria, glioblastoma.
1. Introduction
Glioblastoma is the most common and most aggressive primary brain malignancy. Even with maximum feasible surgical resection with radiotherapy and adjuvant temozolomide (TMZ), survival rates are at a median of 14.6 months from diagnosis in molecularly unselected patients . Radiotherapy and TMZ provides better survival outcomes than radiotherapy alone to treat glioblastoma . Both extent of resection, residual volume are significantly associated with survival and recurrence . Gross total resection is associated with survival improvement, but it is not always possible because the preservation of neurological functions is necessary. The current multimodality treatments including surgery, radiotherapy, chemotherapy for this tumor are still not completely satisfying. Phenethyl isothiocyanate (PEITC) is one of the most extensively studied isothiocyanates . Its effective chemopreventive activity for various tumors and no obvious toxicity in animal models has been reported . PEITC can induce cell cycle arrest and apoptotic cell death . PEITC selectively kills cancer cells, but not normal cells, by generating reactive oxygen species (ROS) to trigger signal transduction, leading to cell cycle arrest and/or apoptosis . Studies have disclosed the cell growth inhibitory effects of PEITC in human leukemia , lung cancer , HeLa cervical cancer , HT-29 colon adenocarcinoma , pancreatic cancer , and human prostate cancer PC-3 cells . In human glioma cells, PEITC inhibits the HIF-1α expression through inhibiting the Phosphoinositide 3-kinase (PI3K) and mitogen activated protein kinase (MAPK) signaling pathway, and reduces the hypoxia-induced secretion of vascular endothelial growth factor (VEGF) ; PEITC also promoted the actions of tumor necrosis factor-related apoptosis-induced ligand (TRAIL) through the upregulation of death receptor-5 (DR5) with ROS-induced-p53 and the
downregulation of cell survival proteins . But the overview of mechanisms of PEITC-induced apoptosis of human brain glioblastoma cells has not been understood well. In the present study, we investigated the effects of PEITC on human brain glioblastoma cells including the induction of cell cycle arrest and apoptosis through the ROS and dysfunction of mitochondria signaling transduction pathways in GBM8401 cells.
2. Materials and methods 2.1. Chemicals and Reagents
PEITC, dimethyl sulfoxide (DMSO), propidium iodide (PI), trypan blue and 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). RPMI-1640, fetal bovine serum (FBS), L-glutamine, penicillin-streptomycin and trypsin-EDTA were purchased from Gibco BRL/Invitrogen (Carlsbad, CA, USA). Primary antibodies (BAX, BCL-2, catalase, Mn-SOD, cytochrome c, caspase-2, -3, -4, -8 and -9, PARP, GRP78, GADD153 and β-actin) and second antibodies for Western blotting were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). They were diluted in PBS Tween-20 before use. Fluo-3/AM, DiOC6 and 4',6-diamidino-2-phenylindole (DAPI) were obtained
from Molecular Probes (Invitrogen, Eugere, OR, USA).
2.2. Cell culture
Human brain glioblastoma multiforme (GBM 8401) cell line was purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were plated onto 75 cm2 tissue culture flasks in RPMI 1640 medium supplemented with
10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mM L-glutamine, and grown at 37°C under a humidified 5% CO2 and 95% air at one atmosphere. The
medium was changed every 2 days .
2.3. Cell morphological changes and viability
GBM 8401 cells (2 × 105 cells/well) onto a 24-well plate were treated with 0, 4, 6,
8, 10, 12, and 14 μM PEITC, or 0, 500 μM TMZ and were incubated for 0, 24 and 48 h. Cells in each well were examined and we took representative photographs at 200× magnification by Nikon TE2000-U inverted microscope for morphological change examinations. After cells from each well were trypanized and collected by centrifugation at 1500 rpm for 5 min, washed twice with PBS, 5 μg/ml PI in PBS was added to the cells to determine the percentage of viable cells. Nonviable cells were stained by PI dye exclusion (indicative of an intact membrane) and displayed brighter fluorescence than the unstained (viable cells). Cells were counted by flow cytometric analysis with FACSCalibur utilizing Cell Quest software (Becton-Dickinson, San Jose, CA, USA) .
2.4. Determination of cell cycle and apoptosis by flow cytometry
GBM 8401 cells (2 × 105 cells/well) were grown in a 24-well plate for 24 h then
treated with 0, 4, 6 and 8 μM PEITC. Cells were isolated, washed with ice-cold PBS, and then fixed in 70% ethanol overnight, followed by the re-suspended in PBS containing 40 μg/ml PI and 0.1 mg/ml RNase and 0.1% Triton X-100 in dark room for 30 min at 25˚C. Cells were washed twice before cell cycle analyses were performed by using a flow cytometer (Beckton-Dickinson) equipped with an argon ion laser at 488 nm wavelength . Fluorescence intensity of the sub-G1 cell fraction represented the apoptotic cell population. Each treatment was performed in triplicate for statistical evaluation.
2.5. Annexin V-FITC/PI double staining
Cell death induced by PEITC was analyzed by flow cytometry. Cell surface exposure of phosphatidylserine (PS) in apoptotic cells was measured by Annexin V-FITC Apoptosis Detection Kit (Biovision, USA). GBM 8401 cells (1×105 cells/well)
were treated with 8 μM PEITC for 24 h and then stained with Annexin V-fluorescein isothiocyanate (FITC) and PI. Cells were washed with PBS after staining, then were re-suspended in binding buffer and immediately analyzed using a fluorescence-activated cell sorting (FACS) flow cytometer (Beckman Coulter, USA) . In fluorescence channels FL1 (488 nm excitation and 530 nm emission for Annexin V-FITC binding) and FL2 (488 nm excitation and red emission for PI), fluorescence from PEITC-treated and untreated cells was detected.
2.6. Detections of reactive oxygen species (ROS), Ca2+ and mitochondrial membrane
potential (ΔΨm) using flow cytometric assay
GBM 8401 cells (2 × 105 cells/well) onto 24-well plates were treated with 0, 8 μM
PEITC or 0, 500 μM TMZ and incubated for 0, 24 and 48 h. Cells were harvested from each treatment by centrifugation, washed twice with PBS and re-suspended in 500 μL of 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) (10 μM) for ROS measurements, in 500μL of Flou-3/AM (2.5μg mL) for Ca2+ level examination and in
500 μL of DiOC6 (200 nmol L) for ΔΨm determination. All samples from control and PEITC treated groups were incubated at 37°C for 30 min in the dark room at 25˚C and analyzed by flow cytometry .
2.7. Measurements of caspase-3, -8 and -9 activities using flow cytometric assay GBM 8401 cells (5×105 cells/well) onto 24-well plate were treated with 0, 8 μM
washed twice with PBS then caspase-3, -8 and -9 substrates (CaspaLux8-L1D2,-CaspaLux 9-M1D2 and PhiPhiLux-G1D2) were added, respectively. Flow cytometric assay was applied to measure the activities of caspase-3, -8 and -9. .
2.8. Western blotting assay
GBM 8401 cells (1 × 106 cells/well) onto 12-well plate were treated with 6, 8 μM
PEITC and incubated for 0, 24 and 48 h to detect the apoptosis-inducing proteins, Cells were collected and lysed in lysate buffer composed of 50 μM tris (pH 8.0), 150 μM NaCl, 5 μM ethylenediaminetetraacetic acid and 0.5% NP-40 with protease inhibitor solution (Roche, Mannheim, Germany). The protein concentration from each treatment was determined by using Bio-Rad protein assay kit. About 30 μg of protein from each sample was separated on a 10% sodium dodecyl sulfate-polyacrylamide electrophoretic gel (SDS-PAGE) and transferred to nitrocellulose membranes (GE Healthcare, NJ, USA). The blot was soaked with blocking buffer of 5% non-fat dry milk in Tris-buffered saline containing Tween-20 (TBS-T) for 1 h at 25˚C. They were incubated with the specific primary antibodies. Immunoreactive proteins were detected with horseradish peroxidase conjugated secondary antibodies and detected by chemiluminescence (GE Healthcare, NJ, USA) and autoradiography by using BioMax LightFilm (Eastman Kodak, New Heaven, CT, USA) .
2.9. Statistical analysis
Results were expressed as mean ± SD in triplicate (n = 3). The differences between PEITC-treated or TMZ-treated and control groups were analyzed by using the Student’s t-test. The p value of less than 0.05 was considered a significant difference. *p<0.05; #p<0.05.
3. Results
3.1. PEITC induced cell morphological changes and decreased the viability of GBM8401 cells
To realize the cytotoxic effects of PEITC, GBM 8401 cells were treated with 0, 4, 6, 8, 10, 12, and 14 μM PEITC for 24 h or treated with 500 μM TMZ for 24 h. Marked morphological changes of GBM 8401 cells based on cells became smaller, round and blunt in size and some floated on medium were induced by PEITC in a dose-dependent manner (Fig. 1A). The total percentages of viable cells were measured by flow cytometric assay, PEITC decreased the percentage of viable GBM 8401 cells in a dose- and time-dependent manner, and TMZ also decreased the percentage of viable GBM 8401 cells (Fig. 1B). The total viable cells were significantly decreased in GBM 8401 cells exposed to more than 6 μM PEITC, and by more than 50% in cells exposed to 10 μM PEITC after 48 h-treatment. Thus the concentration of 8 μM was selected in further experiments.
3.2. PEITC induced sub-G1 phase in GBM 8401 cells
The distribution of cell cycle was examined by flow cytometry after incubation with PEITC for 48 h to further examine the effects of PEITC on the cell numbers of GBM 8401 (Fig. 2A and B). The accumulation of the DNA contents of the GBM 8401 cells exposed to 8 μM of PEITC was significantly increased up to 25% in the G0/G1 phase in contrast to the control (Fig. 2A). The accumulation of sub-G1 phase of the cells exposed to 4, 6, 8 μM of PEITC was significantly increased in contrast to the control (Fig. 2B). Then these cells were analyzed after Annexin-V/PI staining by flow cytometry to confirm the induction of sub-G1 phase of GBM 8401 cells exposed to PEITC (Fig. 2C). Results indicated that PEITC significantly induced apoptosis in GBM 8401 cells.
3.3. PEITC promoted ROS and Ca2+ release and attenuated the levels of
mitochondria membrane potential (ΔΨm) in GBM 8401 cells
Whether the induction of apoptosis in GBM 8401 cells by PEITC through ROS and Ca2+ productions involved the levels of ΔΨm were investigated in further
experiments. ROS productions were increased after GBM 8401 cells exposed to 8 μM of PEITC for 24, 48 h, but not after cells exposed to 500μM of PEITC (Fig. 3A). The levels of ΔΨm were decreased after GBM 8401 cells were treated with PEITC and TMZ (Fig. 3B). PEITC increased Ca2+ release in GBM 8401 cells, but TMZ decreased
it (Fig. 3C). These PEITC effects were in a time-dependent manner. These results indicated that PEITC induced growth inhibition through the ROS production, Ca2+
release, and involved with mitochondria in GBM 8401 cells.
3.4. PEITC induced caspase-8 and -9 activities in GBM 8401 cells
GBM 8401 cells were treated with or without PEITC, TMZ before cells were harvested, and the activities of caspase-8, -9 and -3 and percentage of viable cells were measured by flow cytometric assay to confirm whether caspases were involved in the apoptosis induced by PEITC or TMZ. Caspase-3 activity was not significantly affected after cells were exposed to 8 μM PEITC for 24, 48 h (Fig. 4A). But caspase-8 and -9 activities were significantly increased after cells were exposed to 8 μM PEITC for 24, 48 h (Fig. 4B and C). Caspase-3, -8 and -9 activities were significantly decreased after cells were exposed to 500 μM TMZ for 24, 48 h (Fig 4A, B and C). Therefore caspase-8 and -9 were involved in apoptosis induced by PEITC in GBM 8401 cells.
PEITC induced apoptosis in GBM 8401 cells disclosed by the results from flow cytometric assay. To investigate protein associated with apoptosis, the proteins levels were examined by Western blotting assay after GBM8401 cells were treated with PEITC (6, 8 μM) (Fig. 5). Anti-apoptotic proteins such as Bcl-2, Mcl-1 and Bcl-xl were decreased (Fig. 5A), pro-apoptotic proteins such as Bax, Bad, Bid and Bak were increased (Fig. 5B), the levels of caspase-3, -9, -8, -2, and -4 (Fig. 5C), FADD, Fas, Fas-L and TRAIL (Fig. 5D), Apaf-1, Endo G, Cyto c, AIF, PARP (Fig. 5E) were enhanced, but XIAP was inhibited (Fig. 5E) in GBM 8401 cells. The protein expression of GADD153, GRP 78 and XBP-1 (Fig. 5F), IRE-1α, Calpain 1 and 2 (Fig. 5G) that are associated with ER stress were promoted in GBM 8401 treated with PEITC. PEITC increased the levels of Mn SOD and Cu/Zn SOD but inhibited catalase and GST that are associated with ROS production in GBM 8401 cells (Fig. 5H).
4. Discussion
The effects of PEITC on human glioma cells have been studied . Our study disclosed that PEITC-induced apoptosis of human brain glioblastoma cells in many aspects. PEITC induced cytotoxic effects on GBM 8401 human glioblastoma multiforme cells in vitro: it changed the morphology and decreased the viability of GBM 8401 cells in a dose- and time-dependent manner (Fig. 1A and B). The current chemotherapy agent for glioblastomas, TMZ also decreased the percentage of viable GBM 8401 cells and showed its cytotoxic effects (Fig. 1B). How did PEITC affect the cell cycle of GBM 8401 cells? PEITC significantly increased the accumulation of the DNA contents of the GBM 8401 cells up to 25% in the G0/G1 phase in contrast to the control (Fig. 2A). It also significantly increased the accumulation of sub-G1 phase (apoptotic population) of the cells in contrast to the control and in a dose-dependent
manner (Fig. 2B). Therefore PEITC significantly induced apoptosis in GBM 8401 cells.
PEITC induced growth inhibition through the ROS production, Ca2+ release, and
involved with mitochondria in GBM 8401 cells (Fig. 3). PEITC increased the levels of Mn SOD and Cu/Zn SOD but inhibited catalase and GST that are associated with ROS production in GBM 8401 cells (Fig. 5H). On the contrary, TMZ didn’t increase ROS production, Ca2+ release, but is involved with mitochondria in GBM 8401 cells
(Fig. 3). TMZ can induce mitochondrial damage, such as the loss of ΔΨm, and lead to a decrease of mitochondrial mass in human glioblastoma U87 MG cells, which suggests that mitochondrial damage might play the role in TMZ-induced cell death . Apoptotic pathways include the extrinsic (cytoplasmic, death receptor) and intrinsic (mitochondrial) pathways . The central effectors of apoptosis are caspases. Cell surface death receptors, such as Fas, tumor necrosis factor receptor, or TRAIL receptors are mediated through the extrinsic pathway. The extrinsic death receptor signaling pathway involving the Fas and FasL activates FADD followed by activation of caspase-8, then either activates caspase-3 directly to cause apoptosis or results in Bad activation to affect pro-apoptotic or anti-apoptotic protein expression before leading to mitochondria-mediated pathway . The caspase-dependent pathway involves the Fas receptor then promotes the activations of initiator caspases (caspase-8 and -9), executioner caspases (caspase-3) for induction of apoptosis . In our study, PEITC promoted FADD, Fas, Fas-L and TRAIL (Fig. 5D); PEITC did not significantly affect caspase-3 activity (Fig. 4A), but increased caspase-8 and -9 activities (Fig. 4B and C); PEITC enhanced the levels of caspase-3, -9, -8, -2, and -4 (Fig. 5C). Consequently, activations of initiator caspases (caspase-8 and -9) and increased expressions of caspase-3, -9, -8, -2, and -4 might involve the PEITC-induced apoptosis of GBM
8401 cells. Nevertheless TMZ significantly decreased caspase-3, -8 and -9 activities (Fig. 4A, B and C). TMZ significantly increased the activity of caspase 3 and caspase 9, but did not change the caspase 8 activity in human glioblastoma multiforme T98G cells (European Collection of Cell Cultures) . Thus TMZ has various effects on caspases or induces apoptosis through different pathways in different glioblastoma cell lines, and personalized treatments for glioblastoma is necessary.
The intrinsic (mitochondrial) pathways result in the release of apoptosis-inducing factor (AIF) or endonuclease G (Endo G) to cause apoptosis . Cytochrome c initiates a caspase cascade by advancing apoptosome formation to provide the expression of apoptotic protease-activating factor-1 (Apaf-1). Our data indicated that PEITC enhanced Endo G, Cyto c, Apaf-1 in GBM 8401 cells (Fig. 5E). PEITC decreased anti-apoptotic proteins such as Bcl-2, Mcl-1 and Bcl-xl (Fig. 5A), increased pro-apoptotic proteins such as Bax, Bad, Bid and Bak (Fig. 5B), but inhibited XIAP (Fig. 5E) that is associated with apoptosis progression in GBM 8401 cells. According to these observations above, we suggest that PEITC induces apoptosis also through intrinsic (mitochondrial) pathways as well as extrinsic (death receptor) ones.
Endoplasmic reticulum (ER) stress has been considered one of the factors to lead to diseases through ER stress-induced apoptosis . In animal knockout models, GADD153 mediates apoptosis induced by ER stress in pancreatic cells and by ischemic stress induced in brain neuronal cells . GADD153 induces the expression of the pro-apoptotic Bcl protein, Bak, and the translocation of Bax from the cytosol to the mitochondria . ER stress markers include GADD153, GRP 78, IRE-1α, XBP-1 . In our study, PEITC promoted the protein expression of GADD153, GRP 78 and XBP-1 (Fig. 5F), IRE-1α, Calpain 1 and 2 (Fig. 5G) that are associated with ER stress in GBM 8401 cells.
5. Conclusions
Our experiments have established that PEITC has potent anti-cancer activity through the induction of apoptosis in GBM 8401 cells, and the possible signaling pathways are summarized (Fig. 6). PEITC induces apoptosis through the extrinsic (death receptor) pathway involving the Fas and Fas-L, then activating FADD followed by activation of caspase-8, then either activating caspase-3 directly or through ROS production, dysfunction of mitochondria, AIF and Endo G release from mitochondria before activating caspase-3 through intrinsic (mitochondrial) pathway in GBM 8401 cells. Taken together, these findings reveal the possible molecular mechanisms and signaling pathways of the anti-cancer properties of PEITC for human brain glioblastoma cells.
Acknowledgements
This work was supported by grant TCVGH-1034902B from Taichung Veterans General Hospital, Taichung, Taiwan
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Figures legends
Figure 1. PEITC and TMZ induced cell morphological changes and decreased the percentage of viable GBM 8401 cells. Cells were treated with different concentrations (4-14 μM) of PEITC for 24 h and cells morphological changes were examined under phase contrast microscope at 200x (A) and cells were harvested to measure the percentage of viable cells by flow cytometric assay (B). The values presented are the mean ± S. D. (n = 3) from three independent experiments. Significantly different from vehicle control treated cells at *p < 0.05. C: control, TMZ: temozolomide.
Figure 2. PEITC induced sub-G1 phase and apoptosis in GBM 8401 cells. Cells were treated with different concentrations (4-8 μM) of PEITC for 48 h, and were harvested to measure the cell cycle distribution as described in Materials and Methods. (A) The percentage of cells in G0/G1, S and G2/M phase of cell cycle. (B) The percentage of cells in sub-G1 phase. (C) The percentage of cell in apoptosis after cells were treated with 8 μM PEITC. The values presented are the mean ± S. D. (n = 3) from three independent experiments. Significantly different from vehicle control treated cells at *p <0.05 . C, Ctl: control.
Figure 3. PEITC affected ROS and Ca2+ production and levels of mitochondria
membrane potential (ΔΨm) in GBM 8401 cells. Cells were treated with or without PEITC (8 μM), TMZ (500 μM) for different time periods before being harvested to measure the ROS production by H2DCF-DA (A), the levels of ΔΨm by DiOC6 (B),
and Ca2+ production by Flou-3/AM (C) were analyzed by flow cytometric assay as
described in Materials and Methods. The values presented are the mean ± S. D. (n = 3) from three independent experiments. Significantly different from vehicle control treated cells at *,#p <0.05. C: control, TMZ: temozolomide.
Figure 4. PEITC induced caspase-3, -8 and -9 activities in GBM 8401 cells. Cells were treated with or without PEITC, TMZ before cells were harvested and activities of caspase-3 (A), -8 (B) and -9 (C) were measured by flow cyomteric assay as described in Materials and Methods. The values presented are the mean ± S. D. (n = 3) from three independent experiments. Significantly different from vehicle control treated cells at *,#p <0.05. C: control, TMZ: temozolomide.
Figure 5. PEITC affected apoptosis associated protein levels in GBM 8401 cells. Cells were treated with 6, 8 μM PEITC respectively for 0, 24 and 48 h and then were harvested to measure the apoptotic associated proteins by Western blotting assay (A: Bcl-2, Mcl-1 and Bcl-x. B: Bax, Bad, Bid and Bak. C: Caspase-3, 9, caspase-8, caspase-2 and caspase-4. D: FADD, Fas, Fas-L and TRAIL. E: Apaf-1, Endo G, cytochrome c, AIF, XIAP and PARP. F: GADD153, GRP 78 and XBP-1. G: IRE-1α, Calpain 1 and 2. H: Catalase, GST, Mn SOD and Cu/Zn SOD) in GBM 8401 cells.
Figure 6. The possible signaling pathways of PEITC induced apoptosis in human glioblastoma multiforme GBM 8401 cells.
